Treatment decisions for individual breast cancer patients are frequently based on the number of axillary lymph nodes involved with disease, estrogen receptor and progesterone receptor status, size of the primary tumor, and stage of disease at diagnosis (Tandon et al., J. Clin. Oncol. 7:1120-1128 (1989)). However, even with this variety of factors, it is not possible to predict accurately the course of disease for all breast cancer patients. There is clearly a need to identify new markers in order to separate patients with good prognosis, who will need no further therapy, from those whose cancer is more likely to recur and who might benefit from more intensive adjuvant treatments.
Some of the more promising candidates for new prognostic factors include proto-oncogenes which may be amplified, overexpressed, mutated or otherwise activated in malignant cells. Alterations of proto-oncogenes have been found in many forms of human tumors (Bishop, Science 235:305-311 (1987); Klein et al., Nature 315:190-195 (1985); Varmus, Ann. Rev. Genet., 18:553-612 (1984)). For one of these potentially transforming genes, the c-erbB-2 (HER-2, neu) gene, amplification was shown to be a strong prognostic factor in primary human breast cancers; patients with amplified c-erbB-2 had shorter disease-free and overall survival than patients with no amplification (Slamon et al., Science 235:177-182 (1987); Varley et al., Oncogene 1:423-439 (1987)).
Expression of the c-erbB-2 oncogene protein itself has also been examined for its prognostic potential in both node-positive and node-negative breast cancer. In node-positive patients already known to be at high risk of recurrence, overexpression of c-erbB-2 at the protein level has been consistently associated with shorter disease-free and overall survival. In node-negative patients, however, where improved prognostic prediction has far greater therapeutic implications, c-erbB-2 receptor expression has failed to predict disease outcome in numerous studies (Slamon et al., Science 235:177-182 (1987); Borg et al., Cancer Res. 50:4332-4337 (1990); Paterson et al., Cancer Res. 51:556-567 (1991)). Hence, a more accurate method for characterizing the biological significance of receptor overexpression in node-negative disease could provide clinical dividends.
Recently, the wild-type c-erbB-2 receptor has been shown to exist in two interconvertible forms, p175 and p185 (Epstein et al., J. Biol. Chem. 265:10746-10751 (1990)). The p175/c-erbB-2 receptor isoform exhibits enhanced in vitro tyrosine kinase activity, receptor-specific phosphotyrosine content, and electrophoretic mobility when compared with the p185 isoform. Using conventional single-step c-erbB-2 immunodetection assays, however, tyrosine-phosphorylated p175 and serine/threonine-phosphorylated p185 may be technically indistinguishable.
Only the activated form of a tyrosine kinase is likely to influence cell growth and differentiation. Clinical inconsistencies regarding the role of c-erbB-2 in breast cancer could reflect heterogeneity of receptor activation in vivo. A simple antibody-based method which would distinguish the kinase-active form of c-erbB-2 (arising, for example, due to autocrine or paracrine loops, or receptor mutations) from the inert kinasein-active configuration could provide valuable biological and clinical information.
Moreover, the pharmaceutical industry is interested in evaluating pharmaceutically useful compounds which act as growth factor agonists or antagonists. Tens of thousands of compounds per year need to be tested in an entry level or "high flux" screening protocol. Out of the thousands of compounds scrutinized, one or two will show some activity in the entry level assay. These compounds are then chosen for further development and testing. Ideally, a screening protocol would be automated to handle many samples at once, and would not use radioisotopes or other chemicals that pose safety or disposal problems. An antibody-based approach to evaluating drug growth factor activities would provide these advantages and offer the added advantage of high selectivity.
Activation-specific receptor antibodies have been isolated by other investigators pursuing a variety of experimental studies. However, these isolations appear to have been at least partly serendipitous, and the procedures employed do not provide a systematic approach to this task. Keating et al. (J. Biol. Chem. 263:12805-12808 (1988)) raised polyclonal antibodies to an unphosphorylated peptide corresponding to residues 934-951 of the C-terminal region of the PDGF receptor and subsequently demonstrated that this antiserum recognized the native (immunoprecipitated) but not denatured (immunoblotted) activated PDGF receptor. Keating concluded that ligand-inducible tyrosine kinase activation is associated with a conformational change in the receptor which somehow enables antibody binding. Similarly, Downing et al. (Mol. Cell. Biol. 11:2489-2495 (1991)) raised polyclonal antibodies to an unphosphorylated juxtamembrane domain sequence (552-574) of the CSF-1 receptor. This antiserum also exhibited specificity for the immunoprecipitated (but not denatured) activated receptor isoform, theoretically consistent with conformational change mediated by phosphatidylinositol 3-kinase binding to this region. Campos-Gonzalez and Glenney (Growth Factors 4:305-316 (1991)) isolated a monoclonal antibody, raised against intact receptor, that recognized both the native and denatured form of the EGF receptor. However, the antiserum also recognized EGF receptors lacking the three major tyrosine autophosphorylation sites while failing to recognize .sup.32 P-labelled tryptic peptides; hence, it was proposed that the antibody recognizes a phosphorylation-dependent conformational change which partially renatures following SDS denaturation for immunoblotting. A similar approach using intact proteins as immunogens for monoclonal antibody production was adopted by Hu et al. (Mol. Cell. Biol. 11:5792-5799 (1991)). These workers developed a wide range of monoclonal antibodies to the retinoblastoma (Rb) gene product, some of which distinguished under- and unphosphorylated isoforms of this protein. Gullick et al. (EMBO J. 4:2869-2877 (1985)) prepared antisera to the autophosphorylation sites of the EGF receptor using unphosphorylated peptides, but the resulting antibodies did not distinguish activated and inactivated receptor isoforms. Antibodies directed against phosphotyrosine have also been used in attempts to distinguish activated and inactivated receptor subtypes (Wang, Mol. Cell. Biol. 5:3640-3643 (1985); Wildenhain et al., Oncogene 5:879-883 (1990)), but such antibodies are not receptor-specific. The resulting data are therefore difficult to interpret: tyrosine-phosphorylated bands with electrophoretic mobility between 160 and 190 kilodaltons, for example, could represent activation of the c-erbB-2 receptor, but could also indicate activation of receptors for epidermal growth factor, fibroblast growth factors or platelet-derived growth factor (compare, for example, the electrophoretic mobility of c-erbB-2 and PDGF receptors in FIGS. 4 and 6, Epstein et al., Cell Growth and Differentiation, 3:157-164 (1992)).